Drug Conjugation via Maleimide–Thiol Chemistry Does Not Affect Targeting Properties of Cysteine-Containing Anti-FGFR1 Peptibodies

With a wide range of available cytotoxic therapeutics, the main focus of current cancer research is to deliver them specifically to the cancer cells, minimizing toxicity against healthy tissues. Targeted therapy utilizes different carriers for cytotoxic drugs, combining a targeting molecule, typically an antibody, and a highly toxic payload. For the effective delivery of such cytotoxic conjugates, a molecular target on the cancer cell is required. Various proteins are exclusively or abundantly expressed in cancer cells, making them a possible target for drug carriers. Fibroblast growth factor receptor 1 (FGFR1) overexpression has been reported in different types of cancer, but no FGFR1-targeting cytotoxic conjugate has been approved for therapy so far. In this study, the FGFR1-targeting peptide previously described in the literature was reformatted into a peptibody–peptide fusion with the fragment crystallizable (Fc) domain of IgG1. PeptibodyC19 can be effectively internalized into FGFR1-overexpressing cells and does not induce cells’ proliferation. The main challenge for its use as a cytotoxic conjugate is a cysteine residue located within the targeting peptide. A standard drug-conjugation strategy based on the maleimide–thiol reaction involves modification of cysteines within the Fc domain hinge region. Applied here, however, may easily result in the modification of the targeting peptide with the drug, limiting its affinity to the target and therefore the potential for specific drug delivery. To investigate if this is the case, we have performed conjugation reactions with different auristatin derivatives (PEGylated and unmodified) under various conditions. By controlling the reduction conditions and the type of cytotoxic payload, different numbers of cysteines were substituted, allowing us to avoid conjugating the drug to the targeting peptide, which could affect its binding to FGFR1. The optimized protocol with PEGylated auristatin yielded doubly substituted peptibodyC19, showing specific cytotoxicity toward the FGFR1-expressing lung cancer cells, with no effect on cells with low FGFR1 levels. Indeed, additional cysteine poses a risk of unwanted modification, but changes in the type of cytotoxic payload and reaction conditions allow the use of standard thiol–maleimide-based conjugation to achieve standard Fc hinge region cysteine modification, analogously to antibody–drug conjugates.


MALDI-MS analysis of peptibody hinge region after IdeZ protease cleavage
We performed proteolytic cleavage of peptibodyC19 and its conjugate. We used the IdeZ protease which specifically hydrolyzes the peptide bond right after the hinge region in the IgG sequence. Such cleavage results in an N-terminal peptide containing the hinge region and Fc domain.
In the case of peptibodyC19, we identified the peptide corresponding to the unmodified hinge region ( Figure SA). In the case of the conjugate, we identified the peptide corresponding to the hinge region coupled with a single PEG4-vcMMAE molecule ( Figure SB).
This result shows that one PEG4-vcMMAE molecule is coupled to Cys residue in the hinge region.

LC-ESI-MS analysis of tryptic fragments of peptibody C19 and its conjugate
We performed tryptic digestion of the peptibodyC19 and its conjugate. Resulting tryptic fragments were analyzed by LC-ESI-MS (Table S1 and Table S2). In both cases, we identified the mass corresponding with AESGDDYCVLVFTDSAWTK peptide (2163.9538 Da with carbamidomethylated Cys, Cmpt B inTable S1 and Table S2) that is N-terminal fragment of peptide C19. This result shows that the second PEG4-vcMMAE molecule is not attached to the N-terminal region of the C19 peptide.

IdeZ cleavage of peptibodyC19 and peptibodyC19-conjugate
PeptibodyC19 and peptibodyC19-conjugate was digested with IdeZ (IgG-specific protease) according to the manufactures' protocol (New England, BioLabs, Ipswich, USA). We extended incubation time from 30 minutes to 1 hour at 37°C.

Trypsin digestion in solution
Preparation of samples for LC-ESI-MS analysis was based on theprotocol from Leize-Wagner group from 2016 (Said et al., 2016). PeptibodyC19 and its conjugate were heated to 40°C for 10 minutes. Next, 1 mM TCEP was added and incubated for 10 minutes at 80°C and cooled down to room temperature. Samples were alkylated with 50mM iodoacetamide at room temperature for 30 minutes. Acetonitryle to final concentration of 10% and 1µg of trypsin in Tris-HCl pH 8.0 with CaCl 2 were added and incubated for 3 hours at room temperature, and another 1µg of trypsin was added afterwards. The reaction was continued overnight at 37°C. The samples were reduced for the second time by addition of 1 mM TCEP and incubated at 56°C for 45 minutes. After that, 40% isopropanol and 1% formic acid were added and samples were incubated at room temperature for 2h.

LC-ESI-MS analysis of peptibody C19 and its conjugate fragments
(20 μL) of digests were loaded via autosampler onto a C18 column enclosed in a thermostatted column oven set to 60 °C (Vanquish Flex UHPLC, Thermo Scientific). Samples were held at 12 °C while queued for injection. The chromatographic method was initiated with 5% ACN with 0.1% formic acid. After a 5 min equilibration, peptides were eluted over a 50 min gradient in which ACN content rose to reach 70%. Prior to the next sample injection, the column was washed for 10 min with 90% ACN, then equilibrated at 5% for 15 min. The eluate was diverted to waste for the first 15 min and final 20 min of the run.
Peptides eluting from the chromatography column were analyzed by UV absorption at 220, 248, 280 nm followed by mass spectrometry on the Bruker Compact QTOF (Bruker Daltonics) operated in the positive ionization mode and calibrated with ESI Tuning Mix (Agilent Technologies). Sample solutions were introduced into the MS at a rate of 250 µl/min. Settings: scan range = 400-3000 m/z; nebulizer = 1.8 bar; dry gas = 220°C and 8.0 l/min; capillary = 4500 V; end plate offset = 500 V; hexapole RF = 300 Vpp. Mass range was 350 to 4000 m/z. The spectra were averaged over 15-60 min collection time and deconvoluted using the Bruker Compass Data Analysis software package.